Hemagglutinin (HA) is a major surface membrane glycoprotein responsible for the binding of influenza virus to sialic acid-containing receptors in target cells. Fusogenic activity is triggered by a pH-dependent conformational change of HA in the acidic milieu of the endosomes. HA is a trimeric protein (Mr 195,600) comprising an ectodomain of identical subunits, each of which contains two polypeptides (HA1 and HA2) linked by a disulfide bond. The conformational and thermal stability of HA purified from influenza strain X31 has been investigated by differential scanning calorimetry (DSC), circular dichroism (CD), fluorescence, and ultracentrifugation. HA was found to have a rosette structure with 6 trimers/rosette (31 S) at pH 7.4 to 5.4 in a mixed buffer containing 50 mM phosphate-50 mM acetate with 100 mM NaCl and 1 mM EDTA. Below pH 5.4, HA preparations were heterogeneous and unstable, and intact influenza virus was found also to be rapidly inactivated at pH < 5.4 by Blumenthal et al. in separate studies. The DSC profiles of HA at pH 7.4 +- 1 % octylglucoside showed three domains with Tm = 66 +- 1 C and overall [Delta H] = 1000 +- 100 kcal/mol even though HA was dissociated to trimers (9.4 S) in the presence of the detergent. This indicates that intermolecular interactions between trimers in the rosette structure contribute little to the thermal unfolding parameters. As the pH was decreased from pH 7.4 to 5.4, the Tm and enthalpic values for thermal unfolding decreased from ca 66.5 to 46.7 deg C and from ca 900 to 230 kcal/mol, respectively. The acid-induced destabilization corresponded to tertiary structure loss, as measured by near UV CD and intrinsic tryptophanyl residue fluorescence. Interestingly, temperature-dependent far UV CD measurements indicated that HA secondary structure was actually stabilized (66 to ca 90 deg C) as the protein was acidified (pH 7 to 5). The proton-induced destabilization of tertiary structure and apparent stabilization of secondary structure are novel features of HA.